Search

International audience; Using sea urchin embryos, we demonstrate that the MEK/MAPK/ERK cascade is essential for the proper progression of the cell cycle. Activation of a limited fraction of MAPK/ERK is required between S-phase and M-phase. Neither DNA replication nor CDK1 activation are impacted by the inhibition of this small active MAPK/ERK fraction. Nonetheless, the chromatin and spindle organisations are profoundly altered. Early morphological disorders induced by the absence of MAPK/ERK activation are correlated with an important inhibition of global protein synthesis and modification in the cyclin B accumulation profile. After appearance of morphological disorders, there is an increase in the level of the inhibitor of protein synthesis, 4E-BP, and, ultimately, an activation of the spindle checkpoint. Altogether, our results suggest that MAPK/ERK activity is required for the synthesis of (a) protein(s) implicated in an early step of chromatin /microtubule attachment. If this MAPK/ERK-dependent step is not achieved, the cell activates a new checkpoint mechanism, involving the reappearance of 4E-BP that maintains a low level of protein translation, thus saving cellular energy.

International audience; Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the "translational control" toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed.

International audience; A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model.

The mRNA’s cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.

The eukaryotic translation initiation factor (eIF) 4F facilitates the recruitment of ribosomes to the mRNA 5′ end. The 4E-BPs are small proteins with hypophosphorylated forms that interact with the cap binding protein eIF4E, preventing its interaction with eIF4G, thereby preventing ribosome interaction with mRNA. In sea urchin, fertilization triggers a rapid rise in protein synthesis. Here, we demonstrate that a 4E-BP homologue exists and is associated with eIF4E in unfertilized eggs. We also show that 4E-BP/eIF4E association diminishes a few minutes following fertilization. This decrease is correlated with a decrease in the total amount of 4E-BP in combination with an increase in the phosphorylation of the protein. We propose that 4E-BP acts as a repressor of protein synthesis in unfertilized sea urchin eggs and that 4E-BP/eIF4E dissociation plays an important role in the rise in protein synthesis that occurs shortly following fertilization.

International audience; The eukaryotic Initiation Factor 2 (eIF2) is a key regulator of protein synthesis in eukaryotic cells, implicated in the initiation step of translation. Fertilization of the sea urchin eggs triggers a rapid increase in protein synthesis activity, which is necessary for the progress into embryonic cell cycles. Here we demonstrate that fertilization triggers eIF2α dephosphorylation, concomitant with an increase in protein synthesis and that induction of the eIF2α phosphorylation is intimately linked with an inhibition of protein synthesis and cell cycle arrest. Using a phospho-mimetic protein microinjected into sea urchin eggs, we showed that dephosphorylation of eIF2α is necessary for protein synthesis activity and cell division progression following fertilization. Our results demonstrate that regulation of eIF2α plays an important role in the protein synthesis rise that occurs during early development following fertilization.

Exogenous β casein, previously phosphorylated in vitro by protein kinase A and casein kinase II, was microinjected into Xenopus oocytes to monitor in vivo protein phosphatase activities. Phosphatase activities were 1.6 and 3.4 fmol/min/oocyte, respectively, for β casein phosphorylated by casein kinase II and β casein phosphorylated by protein kinase A. Progesterone induced an early decrease (35% after 10 min) in phosphatase activity restricted to the protein kinase A sites of β casein.

Elongation factor 2 (eEF2) is the main regulator of peptide chain elongation in eukaryotic cells. Using sea urchin eggs and early embryos, two isoforms of eEF2 of respectively 80 and 83kDa apparent molecular weight have been discovered. Both isoforms were identified by immunological analysis as well as mass spectrometry, and appeared to originate from a unique post-translationally modified protein. Accompanying the net increase in protein synthesis that occurs in early development, both eEF2 isoforms underwent dephosphorylation in the 15min period following fertilization, in accordance with the active role of dephosphorylated eEF2 in regulation of protein synthesis. After initial dephosphorylation, the major 83kDa isoform remained dephosphorylated while the 80kDa isoform was progressively re-phosphorylated in a cell-cycle dependent fashion. In vivo inhibition of phosphorylation of the 80kDa isoform impaired the completion of the first cell cycle of early development implicating the involvement of eEF2 phosphorylation in the exit from mitosis.